This application is in response to the Funding Opportunity Announcement PAR-13-253 ?Resource-Related Research Projects for Development of Animal Models and Related Materials (R24)?. We have recently developed a powerful new approach, ?CRISPR-activation (CRISPR-a)?, that allows the over-expression of endogenous genes in a stage- and tissue-specific manner in Drosophila. Here, we propose to build a resource of Drosophila transgenic lines that will allow the systematic analysis of the phenotypes associated with overexpression of fly genes orthologous to genes known or suspected to be associated with human diseases. Genetic model systems such as Drosophila have been used extensively to model human diseases associated with reduction or loss of gene function. By contrast, diseases associated with gene amplification, micro-duplication, or other changes resulting in over-expression have been more challenging to model. Over-expression models would be extremely valuable, as they would provide for example a system for relatively rapid identification of genetic or chemical modifiers of associated phenotypes. To fill this gap, we propose to:
Aim 1. Develop CRISPR-a model flies for diseases associated with gene amplification or duplication. We have identified 279 Drosophila genes that are orthologous to genes amplified in various cancers or other diseases, or duplicated in syndromes associated with chromosomal microduplications. For each, we will generate a corresponding fly stock with two sgRNAs that can be combined with an activating form of Cas9. Additional candidates will be solicited from relevant labs, clinicians and coordinated efforts throughout the grant period;
Aim 2. Develop CRISPR-a model flies for conserved rate- limiting enzymes. Disruption of one or more metabolic pathways is a direct cause of many diseases, and has important impact on many diseases and/or their treatments. The most impactful disruptions to a metabolic pathway occur when a genetic or other perturbation affects the levels of a rate-limiting enzyme. We have identified 300 fly genes orthologous to 390 human genes encoding rate-limiting components of metabolic pathways and will generate transgenic lines for overexpression.
Aim 3. Perform an initial characterization of over-expression models. To increase the value of the resource to the community, we will systematically evaluate the gross phenotypes associated with overexpression of the lines created in Aims 1 and 2. Results will be collected on our existing RSVP phenotype database, which will facilitate search and view of the data by the community.
This project is to build a resource of Drosophila transgenic lines that will allow the systematic analysis of the phenotypes associated with overexpression of fly genes orthologous to genes known or suspected to be associated with human diseases.
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| Ewen-Campen, Ben; Yang-Zhou, Donghui; Fernandes, Vitória R et al. (2017) Optimized strategy for in vivo Cas9-activation in Drosophila. Proc Natl Acad Sci U S A 114:9409-9414 |
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| Flockhart, Ian T; Booker, Matthew; Hu, Yanhui et al. (2012) FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update. Nucleic Acids Res 40:D715-9 |
